Solution structure of omega-conotoxin MVIIC, a high affinity ligand of P-type calcium channels, using 1H NMR spectroscopy and complete relaxation matrix analysis.

We have determined the solution structure of the omega-conotoxin MVIIC from Conus magus by 1H NMR. This conopeptide preferentially blocks P and Q type Ca2+ currents by binding with high affinity to voltage-sensitive Ca2+ channels in neurons. This 26 residue peptide with three disulfide bonds was chemically synthesized and refolded for NMR structural studies. The 1H NMR NOESY spectrum of this peptide was completely assigned, with stereospecific assignments made for 12 of the beta prochiral centers. Complete relaxation matrix analysis using MARDIGRAS was used to obtain initial interproton distances from peak intensities. The correlation time necessary for these calculations was determined by measuring 13C relaxation times using inversely detected natural abundance spectra. Distances were input to DG, which provided 15 starting structures which were then subjected to restrained molecular dynamics calculations using SANDER with the AMBER 91 force field in vacuo. 1H-1H vicinal coupling constants were obtained using a combination of line fitting of both E. COSY and NOESY spectra and used to generate angle restraints that were included explicitly in the restrained molecular dynamics calculations. The final set of the 15 best structures had a backbone rmsd of 0.84 A. The ensemble R1/6 factor calculated by CORMA for the final 15 structures was 11%. The final structure consists of an anti-parallel, triple-stranded beta-sheet, with four turns. In spite of significant differences in amino acid sequence and affinities for calcium channel subtypes, the backbone structure of omega-conotoxin MVIIC is very similar to the previously reported structure of omega-conotoxin GVIA.

Solution structure of omega-conotoxin MVIIA using 2D NMR spectroscopy.

The solution structure of omega-conotoxin MVIIA (SNX-111), a peptide toxin from the fish hunting cone snail Conus magus and a high-affinity blocker of N-type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 20-21, and 24-25. The structure of this toxin is very similar to that of omega-conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.

Proton nuclear magnetic resonance assignments.

The procedures outlined here have been used successfully for more than 30 proteins to date, and are nearly routine for molecules up to a molecular weight of 10,000. Some of the proteins assigned have a molecular weight greater than 10,000. For these larger proteins, relayed-COSY and TOCSY experiments have been essential for the identification of spin systems, although for thioredoxin these experiments could not be used. In this case, assignments were accomplished using nonspecific deuteration to the level of 75% and specific, nearly complete, deuteration of certain kinds of residues (see LeMaster [2], this volume). Nonspecific deuteration reduces the cross-relaxation rates of each proton to the rest of the molecule, thus reducing the linewidths. The cross-peak patterns were also narrowed due to simplification of the coupling patterns. Such a laborious procedure of nonspecific deuteration may not be necessary for complete proton assignments of proteins in this size range, as evidenced by the fact that this method was not used for the other two molecules mentioned above. It may prove, however, to be quite valuable in the study of larger molecules, where linewidths are expected to increase due to longer rotational correlation times. Overlap problems in the NH chemical shifts can be dealt with by making use of the differential temperature dependence of these shifts. Another technique is to take advantage of the wide range of exchange rates between these protons and the solvent. Spectra containing only the slowly exchanging NH protons can be obtained by acquiring spectra of the protein soon after dilution in D2O, and spectra of only the rapidly exchanging protons can be obtained by obtaining spectra in a freshly prepared H2O solution of the protein after having completely exchanged all the NH protons with deuterium. Variation of the pH will resolve problems of overlap in all regions of the spectrum, although many chemical shifts may be unaffected by pH. In some cases, pH variation may change the conformation of the molecule. This may, in fact, assist in the sequential assignment if the chemical shifts can be followed with pH. Finally, the relayed-NOESY experiments can resolve overlap problems with the alpha-proton chemical shifts. Thus, it is very likely that the assignment methods outlined here will be successful for the assignment of the proton spectra of even larger molecules if there is significant secondary structure and significant variety of residues to provide enough dispersion of the chemical shifts.(ABSTRACT TRUNCATED AT 400 WORDS)

NTP and PCr responses to hypoxia by hypothermic and normothermic respiring, superfused, neonatal rat cerebrocortical slices: an NMR spectroscopy study at 14.1 Tesla.

Although mechanisms of hypothermic neuroprotection during oxygen deprivation have long been investigated, further characterizations of various molecular mechanisms are appropriate. Anticipating future studies of hypothermia and hypoxia/ischemia, we investigated the extent to which our ex vivo, NMR-based, superfused brain slice model might be helpful. (Slices are approximately 350 microm thick, with 18 slices per 8 mm NMR tube.) 31P NMR spectroscopic measurements were made of hypothermia-induced changes in high energy phosphates, while simultaneously monitoring and controlling tissue temperature, using 1H NMR, the high spectroscopic resolution available at 14.1 Tesla (600 MHz for protons), and a recently published protocol. NTP and PCr concentrations in healthy, well-oxygenated slices decreased to (55 +/- 15)% and (66 +/- 30)% of their respective values at 28.0 degrees C when warmed to 38.0 degrees C, in approximate agreement with earlier in vivo studies by others. During 30 min hypoxia NTP and PCr decreased to non-observable values, regardless of temperature. After reoxygenation, NTP and PCr recoveries as percentages of respective prehypoxia values were (63% +/- 16%; 70%) +/- 5%) for hypothermic slices (28.0 degrees C), and (46% +/- 13%; 41% +/- hypothermic neuroprotection during oxygen deprivation in this model, which appears suitable for use in further studies.

Akt phosphorylation and cell survival after hypoxia-induced cytochrome c release in superfused respiring neonatal rat cerebrocortical slices.

Phosphorylation of Akt before hypoxia (30 min) and during reoxygenation (4 h) was evaluated in superperfused neonatal rat cerebrocortical slices (350 microm, P7, Sprague-Dawley). Cytosolic cytochrome c intensities in Western blots, which were increased at the end of hypoxia. were decreased during reoxygenation. Western blot intensities of phosphorylated Akt (phospho-Akt), nearly undetectable at the end of hypoxia, recovered quickly during reoxygenation, in a trend opposite that for cytochrome c. At 1.5 h and 4 h after hypoxia they became larger or the same as before hypoxia. Total Akt was unchanged by hypoxia and reoxygenation. Phosphocreatine (PCr) and nucleotide triphosphates (NTP) were measured in parallel 14.1 Tesla ex vivo 31P NMR superfused brain slice studies. PCr and alpha-NTP were nearly undetectable at the end of hypoxia. Although they recovered quickly after hypoxia, they were lower than before hypoxia. Reductions in phospho-Akt during hypoxia were consistent with the general unavailability of basic high energy phosphates. Preferential phosphorylation of Akt after hypoxia suggested that substantial reductions in intracellular energy, as indicated by PCr and NTP, might be tolerated by processes important for generating phospho-Akt. Additionally, the post-hypoxia increase in phospho-Akt might have contributed to concomitant reductions in cytosolic cytochrome c.

Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment.

Complete sequence-specific assignments of the 1H NMR spectrum of bungarotoxin were reported in the previous paper [Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., & Kuntz, I.D. (1988) Biochemistry (first paper of three in this issue)]. The assignment was significantly aided by the use of the homonuclear Hartman-Hahn relayed coherence transfer nuclear Overhauser enhancement spectroscopy experiment (HRNOESY) which we present here, as a modification of relayed coherence transfer nuclear Overhauser enhancement spectroscopy (relayed NOESY) [Wagner, G. (1984) J. Magn. Reson. 57, 497]. As shown here, HRNOESY resolves problems of proton resonance overlap especially in extended chain conformations as found in beta-sheets.

Protein solution structure determination using distances from two-dimensional nuclear Overhauser effect experiments: effect of approximations on the accuracy of derived structures.

Solution structures for many proteins have been determined to date utilizing interproton distance constraints estimated from two-dimensional nuclear Overhauser effect (2D NOE) spectra. Although the simple isolated spin pair approximation (ISPA) generally used can result in systematic errors in distances, the large number of constraints enables protein structure to be defined with reasonably high resolution. Effects of these systematic errors on the resulting protein structure are examined. Iterative relaxation matrix calculations, which account for dipolar interactions between all protons in a molecule, can accurately determine internuclear distances with little or no a priori knowledge of the molecular structure. The value of this additional complexity is also addressed. To assess these distance determination methods, hypothetical "experimental" data, including random noise and peak overlap, are calculated for an arbitrary "true" protein structure. Three methods of obtaining distance constraints from 2D NOE peak intensities are examined: one entails a conservative use of ISPA, one assumes the ISPA to be fairly accurate, and one utilizes an iterative relaxation matrix method called MARDIGRAS (matrix analysis of relaxation for discerning the geometry of an aqueous structure), developed in this laboratory. A distance geometry algorithm was used to generate a family of structures for each distance set. The quality of the average structure from each family was good. The root-mean-square deviation of that average structure from the true structure was improved about 2-5% using the more restrictive rather than the more conservative ISPA approach. Use of MARDIGRAS in a conservative fashion--i.e., with a poor initial model--resulted in improvement in the root-mean-square deviation by 8-15%. With a better initial model, MARDIGRAS obtained even more accurate distances. MARDIGRAS also permits analysis of 2D NOE data at longer mixing times, yielding additional distances. Use of more restrictive ISPA distances did, however, result in a few systematically incorrect structural features in local regions of the protein, producing distortions of 2-3 A. Comparison between experimental data and spectra calculated for the structures correlates with root-mean-square deviation, offering a method of structure evaluation. An R factor for evaluating fit between experimental and calculated 2D NOE intensities is proposed.

Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis.

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold increase in K(m) for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)'s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.

NMR solution structure of an alpha-bungarotoxin/nicotinic receptor peptide complex.

We report the two-dimensional nuclear magnetic resonance (NMR) characterization of the stoichiometric complex formed between the snake venom-derived long alpha-neurotoxin, alpha-bungarotoxin (BGTX), and a synthetic dodecapeptide (alpha 185-196) corresponding to a functionally important region on the alpha-subunit of the nicotinic acetylcholine receptor (nAChR) obtained from Torpedo californica electric organ tissue. BGTX has been widely used as the classic nicotinic competitive antagonist for the skeletal muscle type of nAChR which is found in the avian, amphibian, and mammalian neuromuscular junction. The receptor dodecapeptide (alpha 185-196) binds BGTX with micromolar affinity and has been shown to represent the major determinant of BGTX binding to the isolated alpha-subunit. Previous studies involving covalent modification of the native nAChR from Torpedo membranes with a variety of affinity reagents indicate that several residues contained within the dodecapeptide sequence (namely, Tyr-190, Cys-192, and Cys-193) apparently contribute directly to the formation of the cholinergic ligand binding site. The NMR-derived solution structure of the BGTX/receptor peptide complex defines a relatively extended conformation for a major segment of the "bound" dodecapeptide. These structural studies also reveal a previously unpredicted receptor binding cleft within BGTX and suggest that BGTX undergoes a conformational change upon peptide binding. If, as we hypothesize, the identified intermolecular contacts in the BGTX/receptor peptide complex describe a portion of the contact zone between BGTX and native receptor, then the structural data would suggest that alpha-subunit residues 186-190 are on the extracellular surface of the receptor.

Sequence-specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained.

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to alpha-lytic protease.

alpha-Lytic protease, a bacterial serine protease of 198 amino acids (19 800 Da), has been used as a model system for studies of catalytic mechanism, structure-function relationships, and more recently for studies of pro region-assisted protein folding. We have assigned the backbones of the enzyme alone, and of its complex with the tetrahedral transition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boro Val, using double- and triple-resonance 3D NMR spectroscopy on uniformly 15N- and 13C/15N-labeled protein. Changes in backbone chemical shifts between the uncomplexed and inhibited form of the protein are correlated with distance from the inhibitor, the displacement of backbone nitrogens, and change in hydrogen bond strength upon inhibitor binding (derived from previously solved crystal structures). A comparison of the solution secondary structure of the uninhibited enzyme with that of the X-ray structure reveals no significant differences. Significant line broadening, indicating intermediate chemical exchange, was observed in many of the active site amides (including three broadened to invisibility), and in a majority of cases the broadening was reversed upon addition of the inhibitor. Implications and possible mechanisms of this line broadening are discussed.

Structural studies of alpha-bungarotoxin. 1. Sequence-specific 1H NMR resonance assignments.

We report the complete sequence-specific assignment of the backbone resonances and most of the side-chain resonances in the 1H NMR spectrum of alpha-bungarotoxin by two-dimensional NMR. Problems with resonance overlap were resolved with the assistance of the HRNOESY experiment described in an accompanying paper [Basus, V.J., & Scheek, R.M. (1988) Biochemistry (second paper of three in this issue)]. Significant differences exist between the solution structure described here and the crystal structure of alpha-bungarotoxin, on the basis of the proton to proton distances obtained by nuclear Overhauser enhancement spectroscopy (NOESY) and the corresponding distances from the X-ray crystal structure [Love, R.A., & Stroud, R.M. (1986) Protein Eng. 1, 37]. These differences include a larger beta-sheet in solution and a different orientation of the invariant tryptophan, Trp-28, making the solution structure more consistent with the crystal structure of the homologous neurotoxin alpha-cobratoxin. Four errors in the order of the amino acids in the primary sequence were indicated by the NMR data. These errors were confirmed by chemical means, as described in an accompanying paper [Kosen, P.A., Finer-Moore, J., McCarthy, M.P., & Basus, V.J. (1988) Biochemistry (third paper of three in this issue)].

Structural studies of alpha-bungarotoxin. 3. Corrections in the primary sequence and X-ray structure and characterization of an isotoxic alpha-bungarotoxin.

The most plausible set of chemical shift assignments for alpha-bungarotoxin as deduced from the combined use of two-dimensional J-correlated and two-dimensional nuclear Overhauser effect 1H nuclear magnetic resonance (NMR) spectroscopy was in conflict with the accepted amino acid sequence between residues 8 and 12 and residues 66 and 70 [Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M., & Kuntz, I. D. (1988) Biochemistry (first paper of three in this issue]). Furthermore, NMR spectra of alpha-bungarotoxin, purified by conventional methods, evidenced a second species at the level of approximately 10% total protein. The minor component was separated from alpha-bungarotoxin by Mono-S (cationic) chromatography. Sequencing of Mono-S-purified alpha-bungarotoxin and one of its tryptic peptides showed that the correct sequence for alpha-bungarotoxin is Ser-Pro-Ile at positions 9-11 and Pro-His-Pro at positions 67-69. The electron density map of alpha-bungarotoxin [Love, R. A., & Stroud, R. M. (1986) Protein Eng. 1, 37] was refined with the new sequence data. Improvements in the structure were found primarily for residues 9-11. Sequence analysis of two overlapping tryptic peptides proved that the minor species differed from alpha-bungarotoxin by replacement of a valine for an alanine at position 31. This new toxin, alpha-bungarotoxin(Val-31), binds to the acetylcholine receptor with an affinity that is comparable to that of alpha-bungarotoxin.

Deriving accurate interproton distances from ROESY spectra with limited knowledge of scalar coupling constants via the CARNIVAL algorithm. An iterative complete-relaxation-matrix approach.

A method (termed CARNIVAL) for accurately determining distances from proton homonuclear rotating-frame Overhauser effect spectroscopy (ROESY) is described. The method entails an iterative calculation of the relaxation matrix using methodology introduced with the MARDIGRAS algorithm for analysis of two-dimensional nuclear Overhauser effect spectra (B. A. Borgias and T. L. James, J. Magn. Reson. 87, 475, 1990). The situation is complicated in the case of ROESY as spectral peak intensities are influenced by resonance offset and contributions from homonuclear Hartmann-Hahn (HOHAHA) transfer if the nuclear spins are related by scalar coupling. The effects of spin-locking field strength on distance determinations and the ensuing distance errors incurred when HOHAHA corrections are made with limited knowledge of scalar (J) coupling information have been evaluated using simulated ROESY intensities with a model peptide structure. It has been demonstrated that accurate distances can be obtained with little or no explicit knowledge of the homonuclear coupling constants over a moderate range of spin-locking field strengths. The CARNIVAL algorithm has been utilized to determine distances in a decapeptide using experimental ROESY data without measured coupling constants.

Measurement of chemical shifts and coupling constants for glutamate and glutamine.

Proton chemical shifts and coupling constants were obtained for glutamate and glutamine in water (D2O) at pH = 6.6. Initial chemical shift and coupling constant values obtained from experimental spectra were refined using a spectral simulation and optimization program to get a complete set of values that could not otherwise be measured directly from the experimental spectra due to strong spin-spin couplings. These values are essential for automated spectral fitting procedures that require a priori information.

Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission.

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.

Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5.

15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome b5 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the known assignments of the diamagnetic protein [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375] and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-1H heteronuclear correlation spectra. Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein. As was observed in rat ferrocytochrome b5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme. Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme. The orientation of the z-component of the susceptibility tensors for the two forms are indistinguishable, while the in-plane components appear to differ by about 6 degrees. Differences in the orientation of the in-plane susceptibility components are undoubtedly due dominantly to the relative axial rotation of the heme of between 5 degrees and 10 degrees indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375; Pochapsky, T. C., Sligar, S. G., McLachlan, S. J., & LaMar, G. N. (1990) J. Am. Chem. Soc. 112, 5258-5263].

Solution structure of omega-conotoxin GVIA using 2-D NMR spectroscopy and relaxation matrix analysis.

We report here the solution structure of omega-conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.

Pseudocontact shifts used in the restraint of the solution structures of electron transfer complexes.

The geometry of the ferricytochrome b5-ferricytochrome c complex has been analysed using long-range interprotein paramagnetic dipolar shifts. Heteronuclear filtered NMR spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states. Because pseudocontact shifts can be observed for protons as much as 20 A from the paramagnetic centre, this approach allows study of electron transfer proteins in fast exchange. Our findings provide the first physical evidence confirming hypotheses presented in previous theoretical studies. This absence of certain predicted shifts that are expected based on the best fit to a static model of the complex suggests that cytochrome b5 is more dynamic in solution than in the crystal, in agreement with molecular dynamics simulations.